Project Summary Our long-term goal is to develop drugs that target the cardiac sarcoplasmic reticulum (SR) Ca release channel (ryanodine receptor, RyR2) for heart failure (HF) and arrhythmia therapy. RyR2 Ca release is a key player in regulating cardiac contraction, electrophysiology, energetics and signaling. Abnormally high diastolic SR Ca ?leak? via RyR2, and reduced SR Ca uptake, conspire to reduce SR Ca content and elevate diastolic [Ca]i, hallmarks of both systolic and diastolic HF. Inappropriately timed SR Ca leak is also arrhythmogenic. Thus RyR2 is widely recognized as a molecular target with excellent therapeutic potential for HF and some arrhythmias. Indeed, some repurposed drugs provide proof-of-principle for this concept. To greatly accelerate discovery of drugs that target the RyR2 leak, we propose the first high-throughput screening (HTS) methods using our well-established FRET-based RyR-targeting system and extensive supporting basic research. Pathology-associated RyR leak is associated with two phenotypic features that are sensitive to RyR conformation ? reduced calmodulin (CaM) binding and increased binding of a biosensor peptide (DPc10). We find that SR Ca leak can be reversed by either forced CaM binding or dantrolene (a drug used for acute RyR1 leak in malignant hyperthermia). Dantrolene is unsuitable for chronic use, so we seek novel drugs that restore normal CaM and DPc10 affinity (RyR conformation) and thus inhibit pathological SR Ca leak. We have established direct FRET-based assays of CaM and DPc10 binding to RyR2, and a novel fluorescence lifetime plate-reader enables the translation of these FRET tools into ultrasensitive assays of RyR conformation and interactions with binding partners, in HTS format. Results from pilot screens demonstrate that we are poised to carry out an explicit drug-discovery campaign to detect pathophysiological RyR2 conformations and identify compounds that restore normal RyR2 conformation and function, thus translating our mechanistic research into therapies. Identification of lead compounds from this HTS platform and medicinal chemistry development of analogues will be focused through secondary screens that measure RyR activity in SR membranes, and cellular toxicity and Ca leak in patient-derived iPSC cardiomyocytes and in animal- derived adult ventricular myocytes (normal and HF). Feasibility is ensured by: (1) a robust and sensitive FRET system to specifically resolve RyR structural changes, (2) demonstrated experience applying this FRET system for RyR1-targeted HTS, (3) a novel high-precision FLT-PR, (4) functional insight from parallel hypothesis- driven mechanistic myocyte and animal studies, and (5) top-notch team of MPIs and collaborators. The central hypothesis ? that binding of CaM and DPc10 to RyR2 are key markers of RyR2 pathology ? will be tested in the following Specific Aims: (1) Screen a collection of 50k-350k compounds, and (2) Determine Hit effects on RyR2-mediated calcium leak in control and HF myocytes. Outcomes will include new HTS assays, a model for large-scale HTS campaigns, and novel compounds that may be developed into new drugs or RyR2 probes.